In a process of manufacturing a display substrate (for example, an active-matrix organic light emitting device (AMOLED) display substrate) by an ink-jet printing method, the pixel definition layer is used for defining a shape of the pixel and storing the ink. In order to prevent the ink from being mixed between adjacent pixels, the surface of the pixel definition layer should have a strong hydrophobic property. At the same time, in order to make the ink in a minor shift (for example, the ink drops on the inner edge of the pixel) roll into the pixel groove, the pixel definition layer includes a lower layer with hydrophilic property, and an upper layer with hydrophobic property. However, during above process, there are interface problems in the two-layer structure (the hydrophilic portion and the hydrophobic portion) of the pixel definition layer due to that the pixel definition layer are prone to stratification, which cannot satisfy the requirements of the actual process.